Molecular and functional analysis identifies ALK-1 as the predominant cause of pulmonary hypertension related to hereditary haemorrhagic telangiectasia.

BACKGROUND: Mutations of the transforming growth factor beta (TGFbeta) receptor components ENDOGLIN and ALK-1 cause the autosomal dominant vascular disorder hereditary haemorrhagic telangiectasia (HHT). Heterozygous mutations of the type II receptor BMPR2 underlie familial primary pulmonary hypertension. OBJECTIVE: To investigate kindreds presenting with both pulmonary hypertension and HHT. METHODS: Probands and families were identified by specialist pulmonary hypertension centres in five countries. DNA sequence analysis of ALK-1, ENDOGLIN, and BMPR2 was undertaken. Cellular localisation was investigated by heterologous overexpression of mutant constructs in both BAEC and HeLa cells. The impact of a novel sequence variant was assessed through comparative analysis and computer modelling. RESULTS: Molecular analysis of 11 probands identified eight missense mutations of ALK-1, one of which was observed in two families. Mutations were located within exons 5 to 10 of the ALK-1 gene. The majority of ALK-1 mutant constructs appeared to be retained within the cell cytoplasm, in the endoplasmic reticulum. A novel GS domain mutation, when overexpressed, reached the cell surface but is predicted to disrupt conformational changes owing to loss of a critical hydrogen bond. Two novel missense mutations were identified in ENDOGLIN. CONCLUSIONS: The association of pulmonary arterial hypertension and HHT identifies an important disease complication and appears most common among subjects with defects in ALK-1 receptor signalling. Future studies should focus on detailed molecular analysis of the common cellular pathways disrupted by mutations of ALK-1 and BMPR2 that cause inherited pulmonary vascular disease.

Cloning of the cDNA for the putative calcium-sensing receptor and its tissue distribution in sea bream (Sparus aurata).

The cDNA for the calcium-sensing receptor (CaSR) gene has been cloned from the marine teleost Sparus aurata, the sea bream. The isolated clones were 3.3 kb long with an open reading frame of 2820 bp, a 5' UTR of 240 bp, and 3' UTR of 248 bp. The gene codes for a mature peptide of 940 amino acids which has three principal domains; the extracellular region is more than half the total protein, there is a seven-transmembrane domain, and there is a short intracellular domain. There is considerable sequence identity, 91%, shared between the CaSR of sea bream and puffer fish but overall similarities with mammalian CaSR peptides vary between 44% for rat and mouse and 48% with human CaSR. Nevertheless, the 18 cysteine residues of the extracellular domain are present in all sequences so far analysed of which 9 form a cysteine-rich region in sea bream similar to mammalian CaSR. The distribution of CaSR in sea bream tissues detected by in situ hybridisation showed gene expression in epithelia associated with ion transport or ion regulation including the hind gut, chloride cells of the gills, operculum, gall bladder, pituitary adenohypophysis, and coronet cells of the saccus vasculosus; this distribution was confirmed by RT-PCR. By in situ hybridisation, CaSR gene expression was also present in olfactory nerves and leucocytes.

Calcium-sensing receptors and parathyroid hormone-related protein in the caudal neurosecretory system of the flounder (Platichthys flesus).

The caudal neurosecretory system of the flounder (Platichthys flesus) has been examined by immunocytochemistry and in situ hybridization for the expression of parathyroid hormone-related protein (PTHrP) and calcium-sensing receptors (CaSR). The N-terminus nucleotide and deduced amino acid sequences of flounder PTHrP were determined and used to prepare oligonucleotide probes and homologous antiserum. The Dahlgren cells of the posterior spinal cord and their axons contained PTHrP protein which was also detected around the capillaries of the urophysis. PTHrP gene expression was abundant in the Dahlgren perikarya and axons in the spinal cord, but it was absent from nerve endings in the urophysis. Calcium-sensing receptor protein was present in the Dahlgren perikarya and axons, also with abundant gene expression, but there was neither protein nor mRNA in the urophysis. There were no apparent differences between freshwater- and seawater-adapted fish in either CaSR or PTHrP expression in the caudal neurosecretory system. These observations suggest that Dahlgren cells produce PTHrP which may be released from axons abutting capillaries in the urophysis. However, the sensing of ionic calcium appears to be confined to the perikarya of the Dahlgren cells in the spinal cord neuropil, suggesting that they are responsive to calcium in the central nervous system rather than the general circulation.

Self-organized criticality and the self-organizing map.

The self-organizing map (SOM), a biologically inspired, learning algorithm from the field of artificial neural networks, is presented as a self-organized critical (SOC) model of the extremal dynamics family. The SOM's ability to converge to an ordered configuration, independent of the initial state, is known and has been demonstrated, in the one-dimensional case. In this ordered configuration it is now indicated by analysis and shown by simulation that the dynamics of the SOM are critical. By viewing the SOM as a SOC system, alternative interpretations of learning, the organized configuration, and the formation of topographic maps can be made.

Self-organization in the one-dimensional SOM with a decreasing neighborhood.

A proof of self-organization for a general, standard, one-dimensional SOM and a monotonically decreasing neighborhood function such that W < or = N, is given, where N is the total number of neurons and W the width of the neighborhood function. It is the generalization of already existing proofs for two specific cases with W > or = N/2. Lebesgue continuity of the distribution of the input is not a requirement. The order p of the SOM, where p = [N/W] + 1, if N mod W not = 0 or else p = NIW, is fundamental to the proof. A total of 2p basic intervals of non-zero probability on the support of the input are sufficient for self-organization. These basic intervals are separated by minimum distances which depend on parameters of the SOM (e.g. gain, neighborhood function). The result gives a relationship which in a practical situation can be used to determine, for a given number of neurons and neighborhood function, the minimum number of discrete data points which can guarantee self-organization.

Cloning of the cDNA for sea bream (Sparus aurata) parathyroid hormone-related protein.

This paper reports cloning of the cDNA for sea bream (Sparus aurata) parathyroid hormone-related protein (PTHrP). The gene codes for a 125-amino acid mature protein with a 35-residue prepeptide. The total gene sequence is 1.8 kb with approximately 75% noncoding. The N-terminus of the protein resembles mammalian and chicken PTHrP peptides with 12 of the first 21 amino acids identical and for which there is homology with mammalian parathyroid hormone. Toward the C-terminus, the nuclear transporter region between residues 79 and 93 in sea bream is 73% homologous to tetrapod PTHrP, and the RNA binding domain, 96-117, is 50% homologous, moreover starting with the conserved lysine and terminating with the lysine/arginine sequence. Sea bream PTHrP differs significantly from mammalian and chicken PTHrP, having a novel 16-amino acid segment between residues 38 and 54 and completely lacking the terminal domain associated in mammals with inhibition of bone matrix lysis. RT-PCR and in situ hybridization of sea bream tissues show that the gene is expressed widely and the results confirm observations of a PTHrP-like factor in sea bream detected with antisera to human PTHrP.

Recombinant hyaluronate associated protein as a protective immunogen against Streptococcus equi and Streptococcus zooepidemicus challenge in mice.

The capsule of Streptococcus equi, the cause of strangles, and Streptococcus zooepidemicus, associated with equine lower airway disease, plays an important role in evasion of phagocytosis by polymorphonuclear leucocytes. It is composed of hyaluronate, making it non-immunogenic. A hyaluronate associated protein (HAP) from S. equisimilis, whose gene has been sequenced [1], was investigated (a) for its presence in S. equi and S. zooepidemicus and (b) as an immunogen able to interfere with capsule structure and protect against experimental challenge of mice. The purified capsule of S. equi contained a protein of similar molecular mass to the S. equisimilis protein (approximately 53 kDa). Polymerase chain reaction (PCR) using primers derived from the published sequence of S. equisimilis HAP yielded a product from S. equi and S. zooepidemicus of the expected size and susceptibility to restriction endonucleases. Subcloning of two large in frame StuI/SspI fragments of the HAP gene from S. equi, approximately equivalent to the two halves of the molecule, into the expression vector pGEX-3X yielded only the carboxy half in the correct orientation. This latter recombinant produced a GST fusion protein (HAP-GST) of the expected size that was affinity purified. Antibodies in rabbit antiserum to the native protein in purified hyaluronate reacted strongly in immunoblots with HAP-GST. Antiserum to HAP-GST, when soaked into filter paper strips, caused a diminution of capsule production by S. equi cultured on blood agar. Antiserum added into fresh rabbit blood was not opsonic for S. equi. Immunization with HAP-GST significantly reduced rhinitis in Balb/C mice challenged nasally with S. equi and significantly increased survival time and clearance of bacteria in CBA/CA mice challenged intraperitoneally with S. zooepidemicus.

Application of aqueous two-phase partition in PEG-phosphate systems. Design and implementation of a prototype process for recovery of bulk protein fractions from waste brewer's yeast.

The practical development of a process exploiting aqueous two-phase partition in PEG/phosphate systems is described for the recovery and partial purification of a bulk protein fraction from waste Brewer's yeast. Inadequacies in current assumptions concerning de novo process design of such systems necessitated the empirical establishment of conditions for the two-step extraction of protein from wet-milled yeast suspensions. Experiments with homogeneous proteins and yeast extracts are presented to illustrate the true influence of phase volume upon primary protein extraction, and of added salts and modified system pH upon selective back extraction. A mechanistic description of physical and chemical factors influential upon selective phase partition in PEG/phosphate systems is discussed.

Orbital leiomyosarcoma after radiation therapy for bilateral retinoblastoma.

Leiomyosarcoma rarely occurs in the orbit and is seldom encountered as a postirradiation sarcoma in any anatomic location. Three patients with bilateral retinoblastoma who had received radiation therapy are known to have orbital leiomyosarcoma develop in their third decade of life. The clinical and pathological data pertaining to two of these patients are given and discussed herein.